Speaker
Description
To meet the clinical need for small diameter vascular graft, we developed a new generation of human tissue-engineered vascular graft (TEVG) entirely made of biological material. Vessels were produced by weaving yarn of Cell-Assembled extracellular Matrix (CAM). Study objectives were: 1) to assess the graft behavior in the arterial circulation of immunosuppressed rats to evaluate its in vivo functionality, 2) to identify non-denaturing conservation and sterilization methods in order to simplify the manufacturing process.
CAM sheets were produced in vitro by human skin fibroblasts after 8 weeks of culture in DMEM/F-12 with 20% FBS and 0.5 mM Na L-ascorbate and devitalized before use (frozen/airdried). Ribbons (35/TEVGs, 2-mm-wide) were assembled using a custom-made circular loom into 8-mm-long TEVGs with an internal diameter of 1.6 mm. Woven grafts were terminally sterilized with Ethylene Oxide (EtO) and implanted into the abdominal aorta of immunosuppressed rats. TEVGs were explanted after up to 12 months, and echo doppler was performed before sacrifice. Hematoxylin & Eosin and Masson Trichrome (MT) & Verhoeff stanning were performed, as well as smooth muscles and endothelial cells immunostaining.
CAM ribbons (5-mm-wide) were conserved in different conditions (-80°C, -20°C, 4°C dry or wet, Room Temperature (RT) dry or wet), and mechanically tested after rehydration after 1 year with a tensile test. CAM ribbons (5-mm-wide) were sterilized using different methods: gamma irradiation at high and low dose rate on dry and wet samples, EtO and supercritical CO2 (scCO2), and compared to control (sterile production). They were subcutaneously implanted in immunodeficient rat for up to 10 months and mechanically tested. HE, MT and Alcian Blue stanning were performed, as well as immunostaining (M1/M2).
Twenty rats were successfully implanted, demonstrating graft implantability and the absence of transmural leakage. After 12 months, US images revealed flow through the graft, and weaving pattern conservation. Preliminary results showed formation of a complete neo-media at 3 months. No or very little cell infiltration in the graft was observed.
After 1 year, only samples conserved dry at 4°C and RT showed a decrease in rehydrated cross section area and an increase in maximum force, reminiscent of the behavior of dry CAM. We believe that those samples had diminished ability to rehydrate, which could be problematic for clinical use. For other groups, no mechanical parameters changed after 1 year. Before implantation, maximum force only decreased for the gamma wet group. In this condition, delamination of ribbons was observed with MT. After implantation, the control group showed a steady max force decrease with time, while the other groups displayed a sharp decrease after 2 months, and then no change. After 10 months, no significant difference in maximum force was observed between groups. Histological analysis is ongoing.
In this study, we showed that our woven TEVG can function for up to a year in the arterial circulation of rats. We also demonstrated the long-term conservation of our CAM in conditions compatible with hospital storage. Finally, multiple methods remain promising options for graft sterilization.
20941818487
